Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3–MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.

尽管花青素广泛分布于高等植物中，但甜菜碱已取代石竹目大多数物种中的花青素。石竹目植物中黄酮醇的积累意味着石竹目中从二氢黄酮醇到花青素的花青素生物合成的后期步骤可能被阻断。从石竹植物中分离和表征功能性二氢黄酮醇 4-还原酶 (DFR) 和花青素合酶 (ANS) 表明由于DFR和ANS的抑制而缺乏花青素。在这项研究中，我们证明了菠菜中DFR和ANS的过表达（SoDFR和SoANS，分别）与编码来自矮牵牛的谷胱甘肽S-转移酶（花青素螯合所需）的PhAN9诱导异位花青素在仙人掌Astrophytum myriostigma的黄色花被片中积累。SoANS的启动子测定表明，拟南芥 MYB 转录因子 PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) 激活了拟南芥叶片中的SoANS启动子。拟南芥转录因子与PhAN9的过表达也诱导了黄色仙人掌花被片中异位花青素的积累。来自产生甜菜碱的石竹的 PAP 同源物不激活ANS的启动子. 对 Caryophyllales PAPs 的深入表征和 R2R3-MYB 结构域中的定点诱变确定了影响 Caryophyllales PAPs 反式激活的氨基酸残基。氨基酸残基的取代恢复了石竹PAPs的反式激活能力。因此，MYB 转录因子功能的丧失可能会抑制参与花青素合成后期的基因的表达，导致产生甜菜碱的石竹目植物中缺乏花青素。